A multi-part research program has been designed to yield information about the regulation of tRNA biosynthesis in E. coli and the biological roles and structures of the metabolically stable E. coli 4.5S and 6S RNAs. tRNA gens showing differential effects of metabolic regulation will be isolated and the associated transcriptional control elements characterized by sequence and functional analyses. The transcriptional properties will be compared in a common plasmid expression vector to gain insight into the bases for the different responses to growth-related control. Unique structural elements implicated in control will be modified by in vitro mutagenesis to produce functionally altered variants for comparative evaluation. A major portion of the program will be concerned with the biochemistry of the metabolically stable E. coli 4.5S and 6S RNAs. Primary emphasis will be on discovering and characterizing the biological functions of these small RNAs. We have shown that the 4.5S RNA is essential for growth and that protein synthesis is seriously compromised in cells unable to produce it. The occurrence of the 6S RNA in a complex resembling the 'signal recognition particle' of animal cells suggests a possible role for this species in protein translocation. The strategy in studying the function of these RNAs will be: 1) to identify and characterize the primary function(s) affected when synthesis of the RNA is blocked selectively, 2) to develop and characterize mutants with altered 4.5S or 6S RNA function and 3) to identify 4.5S and 6S RNA binding proteins and characterize their synthesis and function. Finally, structural studies of the 4.5S and 6S RNAs will be initiated to prepare for eventual structure-function studies and in recognition of the need to solve new RNA structures. The existence of recombinant clones which overproduce these RNAs by 30-50 fold makes detailed biophysical characterization possible. First results with the 4.5S RNA indicate that its secondary structure may be a near-perfect hairpin helix. The planned structural analyses, many to be performed in collaboration with others, will include: a) S1 nuclease mapping, b) high resolution proton magnetic resonance spectroscopy, c) laser light scattering, d) microcalorimetry, e) circular dichroism and f) x-ray crystallography.